Extended Data Fig. 1: Functional validation of shRNA screen hits including Gstt1 in metastatic cells.
From: The glutathione S-transferase Gstt1 drives survival and dissemination in metastases
(A) Top 17 shRNA screen hits (minus GSTT1, n = 16) were analyzed for impact on overall survival in pancreatic cancer (KMPlot) based on mean expression of selected genes (N = 177 samples). A long-rank test was used to determine significance (*P = 0.0369). (B) Schematic depicting PDAC mouse models used for obtaining YFP/GFP+ primary and metastatic cells. Created with Biorender.com. (C) Representative fluorescence-activated cell sorting (FACS) strategy to obtain YFP/GFP+ metastatic cells. (D) Expression of Top 17 hits in primary matched GFP+ sorted cells represented as CPM values. Genes significantly differentially expressed between metastases (n = 5) and primary tumors (n = 4) from both mouse models displayed with red asterisk. Two-sided t-test was used to determine statistical significance between primary tumors and metastases (Nr1h3, *P = 0.0220, Gstm1, *P = 0.0496, Itih4, *P = 0.050). (E) qRT-PCR expression analysis of top 6 gene targets in primary (n = 3) and matched metastatic (n = 3) PDAC cell lines. Data represented as metastatic cell line mRNA expression relative to primary tumor-derived cells, s.e.m. Two-way ANOVA was used to determine statistical significance between groups (**P = 0.0065). (F) qRT-PCR for individual shRNA gene knockdowns. Data represents gene expression in a single cell line with n = 2 technical replicates. (G) Individual metastatic cell lines (n = 3) were generated to express each shRNA once and subjected to soft agar assay in (G) for n = 3 replicates. Brightfield images of soft agar colonies from liver metastatic cell lines using individual shRNAs validating top 6 screen hits. (H) Soft agar growth using two independent shRNAs per each gene target. Gene knockdown validation was performed once with three technical replicates for each gene. Data are represented as percent growth relative to NT Control, s.e.m. Two-sided t-test was used to determine statistical significance between groups (*P = 0.0264, **P = 0.0031, **P = 0.0083, **P = 0.0033, **P = 0.0093, *P = 0.0199, *P = 0.0480). (I) Western blot depicting PDAC-derived primary and metastatic cell lines stably expressing control or two independent Gstt1 shRNAs. (J) Soft agar assay growth in matched primary and metastatic cell lines. Representative bright-field image (2.5X). (K) Quantification of soft agar growth. Data are represented as number of colonies per well relative to control. The experiment was performed in triplicate with three technical replicates each. Data are represented as mean s.d. Two-sided t-test was used to determine statistical significance between groups (****P < 0.0001). (L) 2D growth curve in metastatic cell lines. The experiment was performed in triplicate with three technical replicates each. Data are represented as mean s.e.m. Two-sided t-test was used to determine statistical significance between groups.
